1.Washadherentcellstwiceinthedishorflaskwithice-coldPBSanddrainoffPBS.Washnon-adherentcellsinPBSandcentrifugeat800to1000rpminatable-topcentrifugefor5minutestopelletthecells. 2.Addice-coldmodifiedRIPAbuffertocells(1mlper107cells/100mmdish/150cm2flask;0.5mlper5x106cells/60mmdish/75cm2flask). 3.Scrapeadherentcellsoffthedishorflaskwitheitherarubberpolicemanoraplasticcellscraperthathasbeencooledinice-colddistilledwater.TransferthecellsUSPensionintoacentrifugetube.Gentlyrockthesuspensiononeitherarockeroranorbitalshakerinthecoldroomfor15minutestolysecells. 4.Centrifugethelysateat14,000xginaprecooledcentrifugefor15minutes.Immediatelytransferthesupernatanttoafreshcentrifugetubeanddiscardthepellet. 5.Dilutethecelllysateatleast1:10beforedeterminingtheproteinconcentrationbecauseoftheinterferenceofthedetergentsinthelysisbufferwiththeCoomassie-basedreagent.Atthisstep,thesamplecanbedividedintoaliquotsandstoredat-20foruptoamonth. TIP:Whenworkingwithlargevolumesofnon-adherentcells,thecellsmaynotbecooledquicklyenoughtomaintaintheactivityoftheproteinbeingstudied.Inthiscase,pourthecellsuspensionintoamixtureofanequalmassof2xPBSandice,thencollectthecellsbycentrifugationandperformthelysisasdescribedabove.PreparationofCellLysate